CN1242058C - Preparation method and application of epidermal stem cell constructed tissue engineering corneal epithelial implant - Google Patents
Preparation method and application of epidermal stem cell constructed tissue engineering corneal epithelial implant Download PDFInfo
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Abstract
The invention discloses a preparation method of an artificial corneal epithelial implant constructed by epidermal stem cells and application of the artificial corneal epithelial implant in transplantation and repair of an ocular surface which is blind due to limbal stem cell defect. The artificial corneal epithelium implant constructed by epidermal stem cells is prepared by aseptically taking adult neck or goat ear marginal skin, separating and purifying epidermal stem cells by using a chunk culture method and a clone screening method, amplifying by using a serum-free culture medium, and performing CK on the obtained stem cells19、P63、integrin-β1Marker protein immunohistochemistry of isoepidermal stem cellsDetecting; inoculating the purified epidermal stem cells on a human amniotic membrane support without epithelial cells, culturing in a serum-free culture medium, and after 21 days of culture, differentiating the epidermal stem cells on the amniotic membrane to form a multiple layer to obtain an epidermal stem cell amniotic membrane graft; observed by a transmission electron microscope, the epidermal stem cells are divided into 5-6 layers of cells; has a similar structure to normal corneal epithelium. The sheep is used as an experimental animal model to perform autologous or allogeneic epidermal stem cell amniotic membrane graft transplantation, and good effects are achieved.
Description
Technical field
The invention belongs to the tissue engineering technique field, being specifically related to a kind of epidermal stem cells with skin source is the preparation method that the constructed tissue engineered cornea epithelial of seed cell is planted sheet, and this epidermal stem cells is made up tissue engineered cornea epithelial plants sheet and be used to transplant the purposes of reparation because of the blind eye table of limbal stem cell deficiency.
Background technology
Normal eye table is covered with well differentiated cornea and conjunctival epithelium, and both are made of two different on morphology epithelioid cells respectively.Conjunctival epithelium is formed by secreting mucinous goblet cell, and the height vascularization.Cornea is made up of not cornified squamous epithelium, keeps eye table integrity and twenty-twenty vision.Corneal epithelial cell derives from cornea stem cell [the Schermer A et al of corneal limbus, 1986.J Cell Biol.103:49-62.Cotsarelis G.1989.Cell, 57:201-209], various clinically heat, chemical burn, Stevens-Johnson syndrome (Stevens-Johnson Syndvome, SJS), eye table scarring pemphigus (Ocular cicatricial pemphigoid.OCP), various surgical operations and the cold treatment carried out at corneal limbus, the wearing and tearing of contact lens, and serious factors such as infectation of bacteria, all can cause cornea and corneal epithelial stem cells damaged.Contiguous conjunctival epithelium will be invaded cornea and be caused chronic inflammatory diseases, the matrix scarization, and blood vessel hyperplasia has a strong impact on eyesight, even blind.Existing clinically many trials for the treatment of serious eye surface diseases by operative therapy.Plant sheet as amnion load corneal epithelial cell and transplant, can effectively improve the eye table and rebuild effect.In recent years, the corneal epithelial stem cells amnion is planted sheet transplanting reconstruction because the eye surface diseases that limbal stem cell deficiency causes also has report (Pellegrini, G, et al 1997.Lancet, 349:990-993 more; Tsai RJE et al.2000; N Engl J Med.343:86-93, Schwab IR et al, 2000, Cornea.19:421426).Yet limbal stem cell transplantation still faces a following difficult problem: 1. donor tissue deficiency during autotransplantation.2. have immunological rejection during heteroplastic transplantation, postoperative need use a large amount of immunosuppressor.The use of a large amount of immunosuppressor can cause the complication of body, has a strong impact on patient's quality of life.Auto corneal stem cell amnion is planted sheet and is transplanted the impaired cornea of reconstruction, though successfully report is arranged, often causes the cornea of patient both sides impaired to SJS and OCP, and the epithelium of autologous cornea stem cell transplantation then can't be carried out.The Japan scholar, with the rabbit is that animal pattern has carried out planting the research that sheet is rebuild impaired cornea with the oral mucous epithelia amnion, attempt to solve this difficult problem, transplant back 10 days result's report but have only, its result shows: the oral mucous epithelia amnion is planted sheet can rebuild corneal epithelium, but long-term transplantation effect remains further to be observed (Nakamura T et al.2003.Invest Ophthalmol Vis Sci; 37:523-533); Yuan etc. (2002) cultivate epiderm skin cell and corneal epithelial cell on amnion altogether, and the table of discovery chrotoplast is expressed corneal epithelium specificity marker protein K after cultivating certain hour on the amnion
3, prompting free list chrotoplast substitutes corneal epithelial cell and makes up artificial cornea, but further tests.The present invention discloses a kind of successful methods that makes up tissue engineered cornea epithelial with skin epidermal stem cell first, and be that laboratory animal is made the limbal stem cell deficiency model with Central Shanxi Plain milk goat, the tissue engineered cornea epithelial that makes up is carried out from body and allotransplantation.After transplanting back eight months, effect is obvious.
Summary of the invention
The objective of the invention is to, provide a kind of and substitute the purposes that limbal stem cell makes up tissue engineered cornea epithelial and is used to rebuild limbal stem cell deficiency eye table with epidermal stem cells, can't carry out the difficult problem that corneal limbal stem cell autograft is transplanted clinically because of the both sides eyes are impaired to solve, be that a kind of employing epidermal stem cells makes up tissue engineered cornea epithelial, the successful methods of treatment limbal stem cell deficiency.
Realize that the technical solution that above-mentioned purpose adopted is, epidermal stem cells makes up the preparation method that tissue engineered cornea epithelial is planted sheet, adopts the epidermal stem cells that derives from the adult skin histology to be prepared, and it is characterized in that, may further comprise the steps:
1. epidermal stem cells derives from the adult skin histology.
Asepticly take about 0.5 * 0.6cm
2The adult neck skin or the Mammals of size (mainly comprise pet, domestic animal and rare wild animal) ear edge skin, with tissue mass cell culture and cloning screens separation and purification epidermal stem cells, increase with serum free medium, and the acquisition stem cell is carried out CK
I9, P63, integrin-β
1Marker protein immunohistochemistry Deng epidermal stem cells detects, and is all positive, proves employed cell tool epidermal stem cells feature.
2. remove epithelial people's amnion support load epidermal stem cells with a kind of, cultivate with serum free medium, first week be submerged culture (37 ℃, 5%CO
2, saturated humidity), changed liquid in per two days; The cultivation of 2-3 week liquid-vapo(u)r interface (37 ℃, 5%CO
2, saturated humidity), change liquid every day.Cultivate after 21 days, epidermal stem cells is differentiated to form multiple layer on amnion, be the epidermal stem cells amnion that can be used for transplanting and plant sheet.The epidermal stem cells amnion of being manufactured is planted sheet through transmission electron microscope observing, and epidermal stem cells is divided into the 5-6 confluent monolayer cells.It is the progenitor cell feature that basal layer cell is column, and cellular form is normal.Iuntercellular has the desmosome structure, and the hemidesmosome structure is arranged between cell and the basement membrane, shows that the epidermal stem cells amnion plants the analog structure that sheet has the normal cornea epithelium.
1) preparation of serum free medium may further comprise the steps:
(1) inoblast conditioned medium
A. the aseptic Central Shanxi Plain milk goat skin of neck of taking is handled skin chunk with 0.25%Trypsin+0.02%EDTA, and 4 ℃ are spent the night, and epidermis is separated with corium;
B. cell between corium and epidermis is rinsed well, corium is cut into small pieces is affixed in the glass dish, every ware 3-4 piece adds substratum: F12+1%BSA+10ng/ml EGF+10 μ g/ml insulin+10ng/ml IGF-1+ penicillin 100u/ml+ Streptomycin sulphate 100 μ g/ml cultivate;
C. after inoblast is paved with plate, go tissue block, with the inoblast had digestive transfer culture, with 1 * 10
5/ ml is seeded in the 50mm plate, cultivates and collects substratum after 48 hours, divided under the condition centrifugal 30 minutes at 4000r/, and the degerming of 0.22um membrane filtration ,-20 ℃ of preservations are standby, are designated as GSFCM;
(2) IGF-1, EGF, the suitable screening of adding concentration of GSFCM
At F12+1～1.2%BSA is to add EGF in the basic medium respectively, IGF-1, the different designs concentration gradient of GSFCM, divide into groups by orthogonal test, cultivate the epidermal stem cells of institute's separation and purification, and carry out cell counting and survey its growth curve, cloning efficiency and morphocytology change, relatively the relative merits of various combination;
(3) determine that at last culture medium prescription is: F12+1～1.2%BSA+20～25%GSFCM+20～25ng/mlEGF+5 μ g/ml insulin+15～20ng/ml IGF-1+0.4～0.6 μ g/ml hydrocortisone+100u/ml penicillin+100 μ g/ml Streptomycin sulphates.
The preparation method of above-mentioned serum free medium has applied for national patent, and number of patent application is: 200410025938.5.
2) people's amnion (Human amniotic membrane, HAM) making of support.
Amnion take from agree through family members and serological reactions such as HIV, hepatitis A, hepatitis B and syphilis be shown as negative cesarean section delivery puerpera's fetal placenta.In aseptic, the fetal membrane capsule that will have amnion is put into the physiological saline that is added with penicillin, Streptomycin sulphate, and the property of pausing is peeled off attached to the amnion on the fetal membrane capsule; Amnion under peeling off is washed repeatedly with physiological saline, thoroughly washed until blood and other dirts; Again with the PBS flushing that contains penicillin, Streptomycin sulphate 3-5 time; Scrape and pausing property of corneal forceps is peeled off spongy layer with cell.The amnion of removing spongy layer is washed with PBS, be divided into several fritters with eye scissors then, be tiled in respectively in the glass dish, add the pancreatin (EDTA that contains 0.05-0.2%) of 0.1-0.4%, remove epithelial cell through digestion in 8-24 hour; To remove epithelial amnion basal surface and upwards be tiled in the glass dish, the square (according to required definite length of side) that cellulose acetate membrane (NC film) is cut into hollow is thereon attached, and upset makes its amnion epithelial surface upwards put into another culture dish then; In thermostat container, placed 30-120 minute, can use.
The tissue engineered cornea epithelial of made of the present invention, it is good to have transparency, good springiness, characteristics such as workable, can be used for transplanting the reparation mankind or animal and show, be used for all obtaining good effect because of the autotransplantation and the allotransplantation of the blind animal model of limbal stem cell deficiency because of limbal stem cell deficiency causes the serious eye that descends or lose one's sight of eyesight.The serum free medium that uses cultivate epidermal stem cells and tissue engineered cornea epithelial, can weaken the immunological memory of seed cell, immunological rejection when reducing allotransplantation, postoperative need not use a large amount of immunosuppressor, widened the present invention's range of application clinically, not only can be used for autotransplantation, also can be used for allotransplantation.Great advantage of the present invention is to enrich, draw materials conveniently in the skin epidermal stem cell source.
Description of drawings
Fig. 1 is the picture of the model of alkali burned sheep eyes before transplanting;
Fig. 2 is the picture of the epidermal stem cells of purifying;
Fig. 3 is the picture of epidermal stem cells normal proliferation and differentiation on amnion;
Fig. 4 is the picture that the epidermal stem cells amnion is planted sheet;
Fig. 5 is that the epidermal stem cells amnion is planted the sheet transmission electron microscope photo;
Fig. 6 is that the epidermal stem cells amnion is planted the picture that sheet is sutured in plant bed;
Fig. 7 transplants 1 good picture of table reconstruction of sheep model after 245 days;
Fig. 8 transplants the good picture of model 2 sheep eye tables reconstruction after 240 days.
Embodiment
Below in conjunction with Central Shanxi Plain milk goat is that experimental model is described further summary of the invention.
1. animal pattern is made: select the adult Central Shanxi Plain milk goat of healthy no eye illness of 3-4 year, intramuscular injection 846 mixture 2.4ml for animals～2.6ml general anesthesia uses iodine disinfection near the eyes.Baoding of lying on one's side, operation hole, shop towel, eyelid left by eye speculum, two antibiosis reason salt solution (sulfuric acid penicillin 2 * 10
5U/L, Streptomycin sulphate 200mg/L) flushing eye table and conjunctival sac, 2% each 5ml of L-Caine E .3%, retrobulbar anaesthesia.Under ophthalmic operating microscope, cut off manadesma under bulbar conjunctiva and the conjunctiva along art cornea edge annular, expose sclera.The corneal epithelial tissue (about deeply 200um) of 2mm in the sclera of the outer 1mm of annular removal corneal limbus and residual conjunctiva and corneal limbus and the corneal limbus.Burn hemostasis.Then, wipe the central cornea epithelium, and burn to corneal stroma and turn white with having dipped in 1N NaOH cotton swab.Use normal saline flushing 5min immediately.Postoperative art eye paraxin eye water droplet eye, twice of every day.Postoperative is observed every day, and detail record conjunctiva inflammation, symblepharon, situations such as corneal stroma dissolving, perforation.Handle 4 weeks of back, symblepharon and ulcer perforation do not take place in anterior corneal surface vascularization, conjunctivaization.And, judge that belonging to limbal stem cell lacks pathological model fully, as the experimental animal model of transplanting (Fig. 1) according to indexs such as corneal opacity degree and surperficial neovascularization, conjunctivaizations (cell impression learn check a cup cell occurs).
2. epidermal stem cells separation amplification is planted sheet making cultivation with amnion: the aseptic Central Shanxi Plain milk goat ear edge skin of taking, wash several times with PBS at aseptic, and crude removal separates skin under anatomical lens with cartilage, skin is cut into 0.1 * 0.1cm
2The fritter of size, be affixed on up in the 3.5cm plastic ware by epidermis side, add an amount of nutrient solution (M199+15～20%NBS+5～8 μ g/ml insulin+0.5 μ g/ml hydrocortisone+100u/ml mycillin+100 μ g/ml Streptomycin sulphates), changed liquid once in two days, there is the epithelioid cell to spread layer growth around the skin histology piece after 3-4 days, the epidermal stem cells clonal growth is arranged on epithelial lining behind the 7-12d, when treating that these clone's diameters reach 100-150 μ m, these clones of picking under anatomical lens, suitably handle with 0.2%Trypsin and 0.02%EDTA, with glass needle (self-control) these cellular segregation and suction are covered with in the 3.5cm plastic ware of gelatin, with the serum free medium that designs voluntarily, when treating that cell proliferation reaches 75% fusion, digest the cultivation of going down to posterity with 0.25%Trypsin+0.02%EDTA.
The making of separation and purification skin epidermal stem cell may further comprise the steps:
1) at first gets a skin, place the physiological saline that contains mycillin, with PBS () flushing that contains mycillin, dehematize dirt and hair;
2) under anatomical lens, skin and subcutis are separated, skin is cut into 0.1～0.2 * 0.1～0.2cm
2The fritter of size is affixed in the glass dish up by epidermis side, and every ware 3-4 piece adds substratum; The prescription of substratum is: Medium199+15～20%NBS (new-born calf serum)+5～8 μ g/ml insulin (Regular Insulin)+0.5 μ g/ml hydrocortisone+100u/ml mycillin+100 μ g/ml Streptomycin sulphates;
3) place CO
2Cultivate in the incubator, the condition in the incubator is 5%CO
2, saturated humidity, was changed liquid once in per two days by 37 ℃;
4) after 3 days-4 days, grow the growth of epithelioid cell's paving stone sample from the tissue block edge, after 7 days-12 days, on epithelioid cell's layer, there is small round cell to assemble and is the growth of clone's sample, when treating that these clone's diameters reach 100-150 μ m, these clones of picking under anatomical lens are that trypsinase and 0.02%EDTA suitably handle with 0.20%Trypsin, with glass needle these cells suctions are covered with in the 3.5cm plastic ware of gelatin, cultivate with serum free medium;
The prescription of serum free medium is: F12+1～1.2%BSA (bovine serum albumin)+20～25%GSFCM+20～25ng/ml EGF+5 μ g/ml insulin+20ng/ml IGF-1+0.4～0.6 μ g/ml hydrocortisone+100u/ml mycillin+100 μ g/ml Streptomycin sulphates.
When 5) treating that cell proliferation reaches 75% fusion, digest with 0.25%Trypsin+0.02%EDTA, the cultivation of going down to posterity, it is frozen with serum free medium Central Shanxi Plain milk goat epidermal stem cells to be passed for 25 generations, and it is frozen that the human epidermal stem cell was passed for 5 generations;
6) the acquisition cell is carried out the detection of the epidermal stem cells molecular marker that current colleague generally acknowledges: CK19, integrin-β
1P
63, integrin-α
6All positive, what promptly prove institute's sorting is the epidermal stem cells of purifying.
The method of above-mentioned separation and purification skin epidermal stem cell, the applicant has applied for national patent, application number is: 200410025939.X.
The prescription of serum free medium is: F12 (Initrogen Corporation Lot1116568)+1～1.2%BSA (bovine serum albumin Sigma, LOT 716H9310)+20～25%GSFCM (goat skin fibroblast conditionalmedium, GSFCM, goat inoblast conditioned medium)+20～25ng/ml EGF (epithelical cell growth factor, Sigma, E-mail:techserv@sial.com, Product Number E 9644)+5 μ g/ml insulin (Regular Insulin)+15～20ng/ml IGF-1 insulin-like growth factor-1, SigmaE-mail:techserv@sial.com, product Number I3769)+0.4～0.6 μ g/ml hydrocortisone+100 μ g/ml mycillins+100 μ g/ml Streptomycin sulphates;
The present invention is seeded in 3-4 on the epithelial human amniotic membrane support for the epidermal stem cells (Fig. 2) of purifying, with serum free medium cultivate (37 ℃, 5%CO
2, saturated humidity).First week be submerged culture (37 ℃, 5%CO
2, saturated humidity), changed liquid in per two days; The cultivation of 2-3 week liquid-vapo(u)r interface (37 ℃, 5%CO
2, saturated humidity), change liquid every day.Epidermal stem cells is normal proliferation and differentiation (Fig. 3) on amnion.Cultivate after 21 days, epidermal stem cells is differentiated to form multiple layer on amnion, be the epidermal stem cells amnion that can be used for transplanting and plant sheet.The epidermal stem cells amnion of being manufactured is planted sheet, and to have transparency good, good springiness, characteristics (Fig. 4) such as workable.Through transmission electron microscope observing, epidermal stem cells is divided into the 5-6 confluent monolayer cells, it is the progenitor cell feature that basal layer cell is column, cellular form is normal, iuntercellular has the desmosome structure, the desmosome structure is arranged between cell and the basilar membrane, show that the epidermal stem cells amnion plants the analog structure (Fig. 5) that sheet has the normal cornea epithelium.
3. transplant: went on a hunger strike one day before the experiment sheep is transplanted.846 mixture 2.4-2.6ml during operation, the intramuscular injection general anesthesia, the Baoding of lying on one's side cuts off eyelash in operation table, iodine disinfection art eye periphery fur, alcohol takes off iodine.Shop sheep eye wound cloth, eye speculum are opened eyelid, two antibiosis reason salt solution (sulfuric acid penicillin 2 * 10
5U/L, Streptomycin sulphate 200mg/L) flushing eye table and conjunctival sac, 2% each 5ml of L-Caine E .3%, retrobulbar anaesthesia eyeball.Cut off bulbar conjunctiva, expose sclera, 0.05% mitomycin cotton bar is put on the subconjunctival sclera and is acted on 5min, washes repeatedly with physiological saline.Wipe out whole cornea and corneal limbus lesion surface, hyperplastic tissue, make as far as possible that corneal stroma recovers transparent, plant bed is smooth, and burn hemostasis, remove extravasated blood fully, physiological saline washes the back repeatedly and prepares to transplant.Before the transplanting, cultured epidermal stem cells amnion is planted sheet use normal saline flushing 3-5 time, each 5min, different former albumen is to planting the influence of sheet in the elimination substratum.The epidermal stem cells amnion is planted the sheet epithelial surface be covered in art portion downwards, four jiaos of amnions are seam four pins earlier, be fixed on the sclera bad if the art eye exposes, should each fixes a lead-in wire in upper and lower rectus place, at least each hour position tubercle is sewed up a pin, and amnion is fixed in art portion (as Fig. 6).Scratch 2-4 vertical incision gently with keratotomy knife on the amnion that secure attachment is good, the size of otch should be not tear amnion degree of being.2mg dexamethasone, the injection down of each hybrid junctions membrane vesicle of 400,000 unit gentamicins.The peaceful collyrium eye droppings of cornea, art eye eyelid resets, and temporarily covers with sterile gauze.Postoperative first week intramuscular injection every day penicillin, Streptomycin sulphate, dexamethasone, postoperative in three weeks every day 0.3% Ofloxacine USP 23, dexamethasone collyrium eye droppings 2 times, and observe on time and take a picture, handle untoward reaction during the utmost point, remove the partial fixing line second weekend.After this drip profit and relax 6 weeks of collyrium to the, routine observation record art eye changing conditions.Have 3/4 experiment sheep eye table reconstruction effect obvious after eight months, wherein have No. 1, No. 2 model sheep eye table has obvious clear area, and constantly enlarges (Fig. 7-8).
Experimental result shows, make up amnion with amnion load skin epidermal stem cell and plant sheet, can effectively rebuild the damaged corneal limbus of stem cell, and rebuild impaired eye and show, in a single day this invention is applied to human clinical ophthalmology and brings light can for countless people because of the limbal stem cell deficiency blinding, and society brings benefit to the mankind.Can be used for pet simultaneously, rare wild animal, precious domestic animal limbal stem cell deficiency eye table is repaired.
Claims (3)
1. the artificial cornea epithelium of an epidermal stem cells structure is planted the preparation method of sheet, adopts the epidermal stem cells that derives from the adult skin histology to be prepared, and it is characterized in that, may further comprise the steps:
1) the aseptic 0.5 * 0.6cm that takes
2The adult neck skin or the Mammals ear edge skin of size with tissue mass cell culture and cloning screens separation and purification epidermal stem cells, increase with serum free medium, and the acquisition stem cell are carried out CK
19, P63, integrin-β
1The marker protein immunohistochemistry of epidermal stem cells detects, and is all positive, proves employed cell tool epidermal stem cells feature;
2) epidermal stem cells with the purifying in 3-4 generation is seeded on epithelial people's amnion support, cultivates with serum free medium, and first week was submerged culture, culture condition: 37 ℃, and 5%CO
2, saturated humidity was changed liquid in per two days; 2-3 week adopts liquid-vapo(u)r interface to cultivate culture condition: 37 ℃, and 5%CO
2, saturated humidity is changed liquid every day; Cultivate after 21 days, epidermal stem cells is differentiated to form multiple layer on amnion, be the epidermal stem cells amnion that can be used for transplanting and plant sheet.
2. the artificial cornea epithelium that epidermal stem cells as claimed in claim 1 makes up is planted the preparation method of sheet, it is characterized in that the preparation of people's amnion support may further comprise the steps:
1) amnion is got the fetal placenta that HIV, hepatitis A, hepatitis B and syphilis serological reaction are shown as negative cesarean section delivery puerpera, in aseptic, the fetal membrane capsule that will have amnion is put into the physiological saline that is added with penicillin, Streptomycin sulphate, and the property of pausing is peeled off attached to the amnion on the fetal membrane capsule;
2) amnion under will peeling off washes repeatedly with physiological saline, is thoroughly washed until blood and other dirts; Again with the PBS flushing that contains penicillin, Streptomycin sulphate 3-5 time; Scrape and pausing property of corneal forceps is peeled off spongy layer with cell;
3) amnion that will remove spongy layer washes with the PBS that contains penicillin, Streptomycin sulphate, be divided into several fritters with eye scissors then, be tiled in respectively in the glass dish, add the pancreatin of 0.1-0.4%, the EDTA that wherein contains 0.05-0.2% removes epithelial cell through digestion in 8-24 hour;
4) will remove epithelial amnion basal surface and upwards be tiled in the glass dish, be that the NC film is cut into the square thereon attached of hollow with cellulose acetate membrane, and upset makes its amnion epithelial surface upwards put into another culture dish then; In thermostat container, placed 30～120 minutes, and promptly can be used for the cultivation that the epidermal stem cells amnion is planted sheet.
3. the artificial cornea epithelium that epidermal stem cells as claimed in claim 1 makes up is planted the preparation method of sheet, it is characterized in that the prescription of described serum free medium is: F12+1～1.2%BSA+20～25%GSFCM+20～25ng/mlEGF+5g/ml insulin+15～20ng/ml IGF-1+0.4～0.6 μ g/ml hydrocortisone+100u/ml penicillin+100 μ g/ml Streptomycin sulphates.
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| CN101757690B (en) * | 2010-02-05 | 2013-04-24 | 陕西瑞盛生物科技有限公司 | Method for preparing tissue engineering cornea |
| CN101757691B (en) * | 2010-02-05 | 2013-04-24 | 陕西瑞盛生物科技有限公司 | Preparation method of tissue engineering cornea |
| CN102367434B (en) * | 2011-06-03 | 2013-06-05 | 中国人民解放军第二军医大学 | Amniotic membrane microcarrier capable of simulating niche microenvironment for growth of epidermal stem cells and skin substitute thereof |
| CN103055348A (en) * | 2011-10-24 | 2013-04-24 | 北京清美联创干细胞科技有限公司 | Preparation method and application of autologous mesenchymal stem cell-loaded human amniotic membrane cornea paster |
| CN103900883B (en) * | 2014-04-10 | 2016-08-31 | 甘肃农业大学 | Cashmere goat skin is for the preparation method of the ultrathin section of transmission electron microscope observing |
| CN104001216B (en) * | 2014-06-04 | 2016-01-27 | 柯亭羽 | The CFU-GM of skin-derived is utilized to prepare the method for organization engineering skin |
| CN104399125B (en) * | 2014-12-01 | 2016-03-16 | 中国人民解放军第三军医大学第三附属医院 | The method that epidermal stem cells breaks up to sweat gland sample epithelial cell |
| CN108567996A (en) * | 2017-03-07 | 2018-09-25 | 武汉北度生物科技有限公司 | A kind of preparation and its application of 3D multilayer structures cell patch |
| UY38427A (en) * | 2018-10-26 | 2020-05-29 | Novartis Ag | METHODS AND COMPOSITIONS FOR EYE CELL THERAPY |
| CN109517784B (en) * | 2018-11-14 | 2020-03-06 | 洛阳师范学院 | Similar corneal epithelial cell, tissue engineered corneal epithelium, preparation and application |
| WO2022272082A1 (en) * | 2021-06-24 | 2022-12-29 | Rvo 2.0, Inc, D/B/A Optics Medical | Corneal onlay medical device |
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